Outline

Nitric oxide (NO) prevents atherogenesis and inflammation in vessel walls by inhibition of cell proliferation and cytokine-induced endothelial expression of adhesion molecules and proinflammatory cytokines. Reduced NO production due to inhibition of either endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS) may therefore reinforce atherosclerosis. Patients with end-stage renal failure show markedly increased mortality due to atherosclerosis. In the present study we tested the hypothesis that uremic toxins are responsible for reduced iNOS expression. Lipopolysaccharide-induced iNOS expression in mononuclear leukocytes was studied using real-time-PCR. iNOS expression in mononuclear leukocytes was blocked by addition of plasma from patients with end-stage renal failure, whereas plasma from healthy controls had no effect. Hemofiltrate obtained from patients with end-stage renal failure was concentrated, fractionated by preparative reversed phase chromatography, analytical reversed phase chromatography in the displacement- and gradient-mode with trifluoroacetic acid as well as triethylammonium acetate as ion-pair reagent. The chromatographic procedures revealed a homogenous fraction that inhibits iNOS expression in mononuclear leukocytes. Using gas-chromatography/mass-spectrometry the inhibitor of lipopolysaccharide-induced iNOS expression was identified to be phenylacetic acid. Plasma concentrations of phenylacetic acid were determined by magnetic nuclear resonance. In healthy control subjects plasma concentrations were below the detection level, whereas patients with end-stage renal failure had a phenylacetic acid concentration of 3.49 Â± 0.33 mmol/l (n=41). It is concluded that accumulation of phenylacetic acid, a metabolite of phenylalanine, in patients with end-stage renal failure inhibits iNOS expression.